agilent pl-sax 1000Å hplc columns and media pl-sax… · agilent pl-sax 1000Å columns and media...
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Agilent PL-SAX 1000Å columns and media are ideal for the anion-exchange HPLC separations of deprotected synthetic oligonucleotides under denaturing conditions.
The strong anion-exchange functionality, covalently linked to a chemically stable polymer, extends the pH range previously available for anion-exchange chromatography. The anion-exchange capacity is independent of pH.
• Separations using denaturing conditions of temperature, organic solvent, and high pH improve chromatography for self-complementary or G-rich sequences that may associate to form aggregates or hairpin structures.
• Excellent chromatographic performance. Small particle, 5 µm material, for high-efficiency separations of n and n-1 sequences.
• Flexibility. Wide range of particle sizes, 5 µm to 30 µm, and column geometries, 2.1 to 100 mm ID, for analysis and scale-up to purification.
• Long column lifetime. The strong anion-exchange functionality, quaternary amine, covalently linked to a polymeric particle, provides a material with exceptional chemical and thermal stability – even with sodium hydroxide eluents.
Oligonucleotides are synthesized on solid phase supports using an automated procedure of sequential addition of one nucleotide residue. At the end of the synthesis, the oligonucleotide remains attached to the support matrix with the nitrogen and oxygen protecting groups left intact. The cleavage from the solid support matrix removes the protecting groups, with the exception of the dimethoxytrityl (DMT) protecting group attached to the 5’-O-oligonucleotide terminus.
This DMT protecting group can be used to perform a first-step separation based on hydrophobicity, separation of the DMT-on from the DMT-off oligonucleotides. After deprotection, removal of the DMT group, high-performance anion-exchange HPLC can be used to resolve the oligonucleotide product from failure sequences, n-1 and n+1, etc. The selectivity and resolution of this separation is illustrated in Figure 1, the separation of a poly-T-oligonucleotide size standard spiked with a 10 mer, 15 mer, 30 mer, and 50 mer.
Agilent PL-SAX 1000Å HPLC Columns and Media
ConditionsColumn PL-SAX 1000Å 8 µm, 4.6 x 50 mm ID
Eluent A 7:93 v/v acetonitrile: 0.1 M TEAA, pH 8.5
Eluent B 7:93 v/v acetonitrile: 0.1 M TEAA, 1 M ammonium chloride, pH 8.5
Gradient 0-40% B in 10 min, followed by 40-70% B in 14 min and 70-100% B in 25 min
Flow rate 1.5 mL/min
Temperature 60 ºC
Figure 1. High-resolution separation of a poly-T-oligonucleotide size standard spiked with a 10 mer, 15 mer, 30 mer, and 50 mer (main peaks).
2
Phosphorothiolated oligonucleotide analogs have an increased resistance to in vivo nuclease degradation and therefore have been investigated as possible candidates for oligonucleotide therapy. The separation of thiolated from partially thiolated forms is extremely difficult using reversed-phase chromatography, but can be done using anion-exchange at high pH.
The use of high pH minimizes the charge effect of the bases, working above their pKs, and eliminates hydrogen bonding and secondary structural effects. Separation is based on the differences in charge of the oxygen and sulfur in the phosphate groups. The retention of the thiolated oligo is greater than the phosphodiester oligonucleotide, Figure 2.
Ordering information
Agilent PL-SAX 1000Å Columns
Column Dimensions
Column Sizes (length)
50 mm 150 mm 250 mm 300 mm
1.0 mm (ID) 1.0 x 50 mm
5 µm PL1351-1502
2.1 mm (ID) 2.1 x 50 mm 2.1 x 150 mm
5 µm PL1951-1502
8 µm PL1951-1802 PL1951-3802
4.6 mm (ID) 4.6 x 50 mm 4.6 x 150 mm 4.6 x 250 mm
5 µm PL1551-1502
8 µm PL1551-1802 PL1551-3802
10 µm PL1551-3102 PL1551-5102
30 µm PL1551-3702 PL1551-5702
7.5 mm (ID) 7.5 x 50 mm 7.5 x 150 mm
8 µm PL1151-1802 PL1151-3802
25 mm (ID) 25 x 50 mm 25 x 150 mm
10 µm PL1251-1102 PL1251-3102
30 µm PL1251-3702
50 mm (ID) 50 x 150 mm
10 µm PL1751-3102
30 µm PL1751-3702
100 mm (ID) 100 x 300 mm
10 µm PL1851-2102
30 µm PL1851-3102
Agilent PL-SAX 1000Å Bulk Media
Dimensions Quantities
10 g 100 g 1 kg
10 µm PL1451-2102 PL1451-4102 PL1451-6102
30 µm PL1451-2702 PL1451-4702 PL1451-6702
Custom Column and Bulk Media Ordering. Columns and bulk media quantities not listed above can be easily ordered:
• For columns, indicate column dimensions, bonded phase type, particle size, and pore size.
• For bulk media, indicate quantity, bonded phase type, particle size, and pore size.
• Please fax your request to (302) 993-5354 or email [email protected].
• Or you can place your column request at www.agilent.com/chem/customlccol and your bulk media request at www.agilent.com/chem/biohplcprep.
ConditionsColumn PL-SAX 1000Å 8 µm, 4.6 x 50 mm ID
Eluent A 1 M sodium hydroxide
Eluent B 1 M sodium hydroxide, 2 M sodium chloride
Gradient 75-100% B for 25 min, followed by 100% B for 15 min
Linear velocity 360 cm/hr
Figure 2. Separation of a residual phosphodiester (1) and a phosphorothiolate (2) 20 mer oligonucleotide.
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Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing,
performance, or use of this material.
Information, descriptions, and specifications in this publication are subject to change without notice.
© Agilent Technologies, Inc., 2011 Published in the USA, July 29, 2011 Publication Number 5990-8200EN
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